1. Field of the Invention
The present invention relates to use of inducible promoters in the production of glycolic acid, by fermentation. The use of inducible promoters leads to a more stable glycolic acid producer strain.
2. Description of Related Art
Glycolic Acid (HOCH2COOH), or glycolate, is the simplest member of the alpha-hydroxy acid family of carboxylic acids. Glycolic acid has dual functionality with both alcohol and moderately strong acid functional groups on a very small molecule. Its properties make it ideal for a broad spectrum of consumer and industrial applications, including use in water well rehabilitation, the leather industry, the oil and gas industry, the laundry and textile industry, and as a component in personal care products.
Glycolic Acid can also be used to produce a variety of polymeric materials, including thermoplastic resins comprising polyglycolic acid. Resins comprising polyglycolic acid have excellent gas barrier properties, and such thermoplastic resins comprising polyglycolic acid may be used to make packaging materials having the same properties (e.g., beverage containers, etc.). The polyester polymers gradually hydrolyze in aqueous environments at controllable rates. This property makes them useful in biomedical applications such as dissolvable sutures and in applications where a controlled release of acid is needed to reduce pH. Currently more than 15,000 tons of glycolic acid are consumed annually in the United states.
Although Glycolic Acid occurs naturally as a trace component in sugarcane, beets, grapes and fruit, it is mainly produced synthetically. Technologies to produce Glycolic Acid are described in the literature or in patent applications. For instance, Mitsui Chemicals, Inc. has described a method for producing the said hydroxycarboxylic acid from an aliphatic polyhydric alcohol having a hydroxyl group at the end by using a microorganism (EP 2 025 759 A1 and EP 2 025 760 A1). This method is a bioconversion as the one described by Michihiko Kataoka in its paper on the production of glycolic acid using ethylene glycol-oxidizing microorganisms (Biosci. Biotechnol. Biochem., 2001).
Glycolic acid is also produced by bioconversion from glycolonitrile using mutant nitrilases with improved nitrilase activity as disclosed by Dupont de Nemours and Co in WO2006/069110 and U.S. Pat. No. 7,445,917. These documents teach a process using formaldehyde and hydrogen cyanide as precursors for the synthesis of glycolonitrile, and using an enzyme catalyst having nitrilase activity for the synthesis of glycolic acid from glycolonitrile. The main disadvantage of this process is that glycolonitrile is a chemical substance which may polymerize violently under the influence of traces of acid, or base, with fire or explosion hazard. This substance decomposes on heating producing toxic fumes including hydrogen cyanide and nitrogen oxides. Therefore it is listed as an extremely hazardous substance.
Methods for producing Glycolic Acid by fermentation from sugar, and in particular from renewable resources, using bacterial strains are disclosed in patent applications from Metabolic Explorer (WO 2007/141316 and WO 2010/108909).
The biological production of glycolic acid requires the formation of intermediates from the central metabolism of the bacterium (see FIG. 1.). Isocitrate situated at the junction of the Krebs cycle and the glyoxylate shunt is one of them (Tricarboxylic acid cycle and glyoxylate bypass, reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik. W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology). Isocitrate is either (1) cleaved into succinate and glyoxylate, a reaction catalyzed by isocitrate lyase, encoded, by the aceA gene or (2) converted into α-ketoglutarate by isocitrate dehydrogenase, encoded by the icd gene. Previous work described in patent application EP 2 027 277 has shown good productions of glycolic acid by strains having an attenuated expression of the icd gene. Reducing the flux in the TCA cycle to force it towards the glyoxylate shunt increased the yield of glycolic acid production significantly but at the same time, it weakened the strain.
The strains with an attenuated expression of the icd gene were not stable when grown for many generations, which is a strong disadvantage for industrial use. The authors found a solution to the problem by using inducible promoters.
Use of inducible promoters in biotechnological processes is in the art of industrial biotechnology. These promoters usually respond to chemical or physical stimuli exemplified by propionate (WO2007005837), zinc (WO2004020640), arabinose (WO1998011231), temperature (‘Microbial conversion of glycerol to 1,3-propanediol by an engineered strain of Escherichia coli.’ Tang X, Tan Y, Zhu H, Zhao K, Shen W. Appl Environ Microbiol. 2009 March; 75 (6): 1628-34.) and light.
Efficient glycolic acid production requires fine tuning of pathways. For maximum glycolic acid production and improved stability of producer strains, it can be beneficial to be able to modulate the expression of certain key enzymes during the process. For instance, the expression of the icd gene is absolutely required for biomass production but not for glycolic acid production and vice versa for aceA. Therefore, use of inducible promoters may be of interest in improving the overall yield of producing glycolic acid at an industrial level.
At this point use of inducible promoters to control expression of genes involved in glycolic acid production has never been considered nor reported.
The inventors have found that heterologous inducible promoters may be beneficial when used to regulate gene expression of genes involved in complex metabolic pathways such as glycolic acid biosynthesis.